Multiprobe laser reflectometry in imaging and characterization of biological tissues

Laser backscattered radiation from human forearm and foot were measured by multi-probe reflectometer, which consisted of one input probe and three output probes placed at distances of 2, 4 and 6 mm from the input probe. The normalized backscattered intensity (NBI) signals from the tissue surface, measured by the output probes, after digitization, were used to reconstruct the reflectance images of tissues in various layers below the skin surface. From NBI profiles measured at various locations of the tissues on the forearm the corresponding optical parameters, the scattering (mu(s)) and absorption (mu(a)) coefficients and the anisotropy parameter g, by matching these with profiles as simulated by Monte Carlo procedure were determined. From these data the optical parametric images of forearm were reconstructed which show the variation of these parameters at various locations. Similarly, the NBI data were collected from the foot sole region of healthy and diabetes subjects and their images reconstructed. These images showed the variation in the NBI in the diabetic foot sole compared to that of healthy subject, indicating the tissue structural changes. These procedures could be useful for diagnostic and therapeutic applications of lasers.     

Considerations of coherent scattering and electron binding in incoherent scattering, in computation of dose deposition in tissue from low-energy photon beams

Two different sets of Monte Carlo computations were carried out for the study of dose penetration of monoenergetic, low-energy (10 to 100 keV) photon beams incident on slabs of tissue. One program took into account coherent scattering and considered electron binding when finding the angle of scattering during incoherent scattering; the other simpler program, customarily used at higher energies, largely ignored these effects. For calculations at the source photon energy of 100 keV, it was found that there was negligible difference in dose distribution in the slab between the more and less complex type of calculations. The same thing was found to be true for the 30 and 10-keV source photon energies only for shallow penetration distances; and at deeper penetrations the simple approach tended to overestimate the dose appreciably. It is concluded that for penetration of low-energy photon beams into tissue, accurate calculational results cannot be assured with the neglect of coherent scattering effects and electron binding considerations in determining the scattering angles except for shallow depths of penetration.     

Image reconstruction of optical attenuation coefficient variation in biological tissues

A procedure for non-invasive imaging of the optical attenuation coefficient variation of in vivo thick organs/tissues is developed. The laser back-scattered surface profiles at various locations of human forearm, by multi-probe reflectometer, are measured. These profiles are matched by iterative procedure, with that as obtained by Monte Carlo simulation and the corresponding values of attenuation coefficient (equal to the sum of absorption and reduced scattering coefficients) are determined. By interpolation of this data a 100 x 100 grid is constructed and after median filtering of this data a color-coded image of the variability of the optical attenuation coefficient of the forearm is obtained. These images in different subjects show variation due to change in overall tissue composition and blood pooling. This non-invasive imaging procedure may help in identifying the diseased affected regions in healthy tissues and in application of photodynamic therapy.     

Initial studies of the application of the linear signal transfer theory in evaluating diaphanoscopic examinations exemplified by rheumatism diagnosis]

Rheumatoid arthritis affecting the small joints--in particular the fingers--has advantageous geometry for the transmission of near-infrared (NIR) light. Examination of the optical properties of tissues has revealed that as a result of changes to the capsule and synovial fluid there is a considerable increase in photon scattering already in the early stages of the disease--in particular around 685 nm. This suggests the appropriateness of analysing the photon density profile resulting from punctiform irradiation of the joint. In a first approximation, the point spread function of transmitted photon density is confirmed to be proportional to a Gauss distribution, as suggested by Arridge. In accordance with the linear signal transfer theory, therefore, it is possible to establish a virtual transfer system described by a first-order differential equation. (The tissue optical conditions mu a < mu's and mu a = constant (mu a = absorption coefficient) were assumed). The parameter mu's (= reduced scattering coefficient) was determined by linear approximation of the Gauss distribution to the calculated or measured point spread function. For selected patient data, the mu's was determined in healthy and diseased finger joints (e.g. 10.1 cm-1 and 26.8 cm-1, respectively), and the results were in good agreement with those obtained experimentally.     

Treatment of mobile phase particulate matter in low-angle quasi-elastic light scattering

The method of quasi-elastic laser light scattering (QLS), particularly at low forward scattering angles, has been complicated by the transient presence of Mie or large Rayleigh scattering particles which contaminate the scattering volume. These large contaminating particles have substantial effects on photon correlation spectroscopy because the presence of these larger scatterers tends to decrease the value of the apparent diffusion coefficient of the particle of interest. A method is presented which yields more accurate diffusion constants by autocorrelation of selected photon count periods representative of minimal Mie or large Rayleigh particle contamination. This method was applied to the determination of the apparent diffusion constant for four proteins—ovalbumin, chymotrypsinogen-A, bovine serum albumin, and ribonuclease-A.     

用蒙特卡罗方法模拟光在多层组织中的吸收特性

在讨论目前新颖的组织功能成像打骂能性（例如光声成像）时,光子在组织中的吸收和散射特性是一个很重要的问题,鉴于这一点,本文利用一个多层模型研究了光子在皮肤,脂肪和肌肉组织中的吸收和散射特性,得到了在组织中某一深度处光子在一个平面上的吸收分布,以及在不同吸收系数和散射系数的情况下,光子的反射,吸收和透射几率,结果表明在经过多次散射后,大部分的光子被吸收,在本文的模型中只有7.3%的光子从表面反射（包括镜面反射和漫反射）,还讨论了不同光学参灵敏对参流分布的影响。     

Dynamic light scattering: a practical guide and applications in biomedical sciences

Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a very powerful tool for studying the diffusion behaviour of macromolecules in solution. The diffusion coefficient, and hence the hydrodynamic radii calculated from it, depends on the size and shape of macromolecules. In this review, we provide evidence of the usefulness of DLS to study the homogeneity of proteins, nucleic acids, and complexes of protein–protein or protein–nucleic acid preparations, as well as to study protein–small molecule interactions. Further, we provide examples of DLS’s application both as a complementary method to analytical ultracentrifugation studies and as a screening tool to validate solution scattering models using determined hydrodynamic radii.     

Quasi-elastic light scattering of Artemia ribosomes sedimented in a CsCl density gradient

It is impossible to measure the diffusion coefficient of macromolecules directly and accurately by quasi—elastic light scattering, when aggregates cannot be eliminated from the solutions to be investigated. Nevertheless, a simple method can be applied to overcome this problem in many cases. Aggregates are separated from the monomeric macromolecules by rate-zonal sedimentation in a CsCl density gradient in a transparent centrifugation tube; the monomers are then located by laser light scattering intensity measurements; photon correlation spectroscopy of the scattered light finally yields their diffusion coefficient. The viscosity of aqueous CsCl solutions at different temperatures and concentrations allows a good separation by centrifugation and a low uncertainty in the reduction of the measured diffusion coefficient to standard conditions.The application of the method to eukaryotic large ribosomal subunits is described as an example.     

SAXS-data-based structural modeling of DNA–gadolinium complexes fixed in particles of cholesteric liquid-crystalline dispersions

Structure of cholesteric liquid-crystalline dispersions (CLCDs) formed by double-stranded DNA molecules and treated with gadolinium salts was studied by small-angle X-ray scattering (SAXS). The obtained SAXS data open the way for structural modeling of these complexes to obtain a reasonable explanation for the correlated decrease in amplitude of an abnormal negative band in the circular dichroism (CD) spectra and the characteristic Bragg peak in the experimental small-angle X-ray scattering curves observed on treatment of CLCD by gadolinium salts. Model simulations of different kinds of structural organizations of the DNA–gadolinium complex were performed using novel SAXS data analysis methods in combination with several new, complementary modeling techniques, enabling us to build low-resolution three-dimensional structural models of DNA–gadolinium complexes fixed in CLCD particles. The obtained models allow us to suggest that a change takes place in the helical twist of quasinematic layers formed by these molecules at high concentrations of gadolinium salt. This change in the twist can be used to explain the experimentally observed increase in amplitude of an abnormal band in the CD spectra of DNA CLCD.     

时间分辨反射确定生物组织光学性质的方法研究

通过考虑吸收系数对漫射常数的影响,改进了时间分辨反射中两点测量生物组织光学性质的方法,对约化散射系数与吸收系数之比较小的生物组织进行了模型测量,结果表明此种不仅减小了测量的系统误差,而且扩展了两点测量法的应用范围。同时也较为详细地讨论了有效时间的选择和光源与探测点间的距离对测量结果的影响。     

Quantum squeezed state analysis of spontaneous ultra weak light photon emission of practitioners of meditation and control subjects

Research on human ultra-weak photon emission (UPE) has suggested a typical human emission anatomic percentage distribution pattern. It was demonstrated that emission intensities are lower in long-term practitioners of meditation as compared to control subjects. The percent contribution of emission from different anatomic locations was not significantly different for meditation practitioners and control subjects. Recently, a procedure was developed to analyze the fluctuations in the signals by measuring probabilities of detecting different numbers of photons in a bin and correct these for background noise. The procedure was tested utilizing the signal from three different body locations of a single subject, demonstrating that probabilities have non-classical features and are well described by the signal in a coherent state from the three body sites. The values indicate that the quantum state of photon emitted by the subject could be a coherent state in the subject being investigated. The objective in the present study was to systematically quantify, in subjects with long-term meditation experience and subjects without this experience, the photon count distribution of 12 different locations. Data show a variation in quantum state parameters within each individual subject as well as variation in quantum state parameters between the groups.     

Tissue-Specific Optical Mapping Models of Swine Atria Informed by Optical Coherence Tomography

Computational models and experimental optical mapping of cardiac electrophysiology serve as powerful tools to investigate the underlying mechanisms of arrhythmias. Modeling can also aid the interpretation of optical mapping signals, which may have different characteristics with respect to the underlying electrophysiological signals they represent. However, despite the prevalence of atrial arrhythmias such as atrial fibrillation, models of optical electrical mapping incorporating realistic structure of the atria are lacking. Therefore, we developed image-based models of atrial tissue using structural information extracted from optical coherence tomography (OCT), which can provide volumetric tissue characteristics in high resolution. OCT volumetric data of four swine atrial tissue samples were used to develop models incorporating tissue geometry, tissue-specific myofiber orientation, and ablation lesion regions. We demonstrated the use of these models through electrophysiology and photon scattering simulations. Changes in transmural electrical conduction were observed with the inclusion of OCT-derived, depth-resolved fiber orientation. Additionally, the amplitude of optical mapping signals were not found to correspond with lesion transmurality because of lesion geometry and electrical propagation occurring beyond excitation light penetration. This work established a framework for the development of tissue-specific models of atrial tissue derived from OCT imaging data, which can be useful in future investigations of electrophysiology and optical mapping signals with respect to realistic atrial tissue structure.     

Diagnosing colorectal abnormalities using scattering coefficient maps acquired from optical coherence tomography

Optical coherence tomography (OCT) has shown potential in differentiating normal colonic mucosa from neoplasia. In this study of 33 fresh human colon specimens, we report the first use of texture features and computer vision-based imaging features acquired from en face scattering coefficient maps to characterize colorectal tissue. En face scattering coefficient maps were generated automatically using a new fast integral imaging algorithm. From these maps, a gray-level cooccurrence matrix algorithm was used to extract texture features, and a scale-invariant feature transform algorithm was used to derive novel computer vision-based features. In total, 25 features were obtained, and the importance of each feature in diagnosis was evaluated using a random forest model. Two classifiers were assessed on two different classification tasks. A support vector machine model was found to be optimal for distinguishing normal from abnormal tissue, with 94.7% sensitivity and 94.0% specificity, while a random forest model performed optimally in further differentiating abnormal tissues (i.e., cancerous tissue and adenomatous polyp) with 86.9% sensitivity and 85.0% specificity. These results demonstrated the potential of using OCT to aid the diagnosis of human colorectal disease.     

High-Resolution Optical Molecular Imaging of Changes in Choline Metabolism in Oral Neoplasia

This study was aimed at developing an optical molecular imaging approach to measure differences in uptake and intracellular retention of choline in clinically isolated tissue biopsies from head and neck cancer patients. An optically detectable analogue of choline (propargyl choline) was synthesized and evaluated in 2D and 3D models and clinically isolated paired biopsies (n = 22 biopsies). Fluorescence contrast between clinically abnormal and normal tissues based on uptake and intracellular retention of propargyl choline was measured and correlated with pathologic diagnosis. Results in 2D and 3D models demonstrated a rapid uptake of propargyl choline in cancer cells, uniform permeation in tissue models, and specific detection of intracellular entrapped propargyl choline using the click chemistry reaction with an azide-modified Alexa 488 dye. Fluorescence imaging measurements following topical delivery of propargyl choline in clinically isolated biopsies showed that the mean fluorescence intensity (MFI) of neoplastic tissues was four-fold to five-fold higher than the MFI of clinically and pathologically normal samples. This difference in fluorescence contrast was measured on the basis of comparison of paired biopsy sets isolated from individual patients as well as comparison of clinically abnormal and normal biopsies independent of anatomic locations in the head and neck cavity and across diverse patients. In conclusion, a novel imaging approach based on monoalkyne-modified choline was developed and validated using cell and tissue models. Results in clinically isolated tissue biopsies demonstrate a significant fluorescent contrast between neoplastic and normal tissues and illustrate high specificity of the optical imaging approach.     

Malachite green and phthalocyanine-silver reactions reveal acidic phospholipid involvement in calcification of porcine aortic valves in rat subdermal model

Subdermal implant models are helpful in the study of calcification "in vivo" and for testing anticalcific treatments. After implantation of porcine aortic valve leaflets in rat subcutis, we previously found that glutaraldehyde-Cuprolinic blue reactions (GA-CB) at low pH induce favourable tissue unmasking from mineral deposits, and visualize peculiar, electrondense layers that outline the calcifying cells and matrix vesicle-like structures. The layer-forming material seemed to consist of acidic phospholipids because of its anionic nature and differential susceptibility to chemical/enzymatic extractivity. In the present investigation, pre-embedding glutaraldehyde-Malachite green (GA-MG) reactions and subsequent osmium post-fixation were compared with pre-embedding GA-CB reactions, combined with post-embedding von Kossa silver staining (GA-CB-S), to assess whether the layer-forming material is actually composed of acidic phospholipids and exhibits calcium-binding properties. After lowering standard pH, GA-MG reactions also caused sample demineralization and the appearance of pericellular osmium-MG-reactive layers comparable to CB-reactive ones. Moreover, GA-CB-S reactions showed that major silver precipitation was superimposed to the CB-reactive layers, whereas minor metal extra-precipitation occurred at three distinct, additional sites. These results demonstrate that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface, replacing cell membrane and acting as major apatite nucleator. However, the overall observations are consistent with the hypothesis that certain phases are common to the various types of normal and/or abnormal calcification.     

Effects of nitrogen deprivation on cell division and expansion in leaves of Ricinus communis L.

The effects of nitrogen deprivation on leaf extension, cell numbers and epidermal cell size were followed in leaves of Ricinus communis L. The extent to which reductions in final cell number or final epidermal cell size contributed to the reduction in final leaf size depended on the developmental stage of the leaf at the time of N deprivation. In leaves which already had their full complement of cells (leaf 2), the reduction in final leaf size following nitrogen deprivation was associated with a reduction in final cell size. In leaves that were at earlier stages of development at the onset of N deprivation (leaves 3 and 4), the reduction in final leaf size was greater than in leaf 2. In these younger leaves, the final cell size was even smaller than in leaf 2, but the greatest contribution to reduced final leaf size was a reduction in the number of cells produced. This accounted for approximately 80% of the reduction in final leaf size in leaf 4. During leaf development, the contribution from different tissue layers to the total cell number changed. In the smallest leaf sizes, the contribution from upper and lower epidermis and spongy parenchyma was greater than that from palisade parenchyma. As the leaf size increased, cells in the palisade parenchyma continued to divide for longer than in the other layers. At final leaf size, the contribution from the different tissue layers to total cell number was the same for leaves 2, 3 and 4, irrespective of N treatment. In these final leaf structures, palisade parenchyma contributed 60% of the total cell number. Thus, although nitrogen deprivation affected leaf size variously through cell division and cell expansion, depending on leaf developmental stage at the time of nitrogen deprivation, the ratio of cell numbers and sizes in different tissue layers, at final leaf size, was unaffected.     

Forced Detection Monte Carlo Algorithms for Accelerated Blood Vessel Image Simulations

Two forced detection (FD) variance reduction Monte Carlo algorithms for image simulations of tissue‐embedded objects with matched refractive index are presented. The principle of the algorithms is to force a fraction of the photon weight to the detector at each and every scattering event. The fractional weight is given by the probability for the photon to reach the detector without further interactions. Two imaging setups are applied to a tissue model including blood vessels, where the FD algorithms produce identical results as traditional brute force simulations, while being accelerated with two orders of magnitude. Extending the methods to include refraction mismatches is discussed. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Quantitation of time- and frequency-resolved optical spectra for the determination of tissue oxygenation.

The recent development of near-infrared time- and frequency-resolved tissue spectroscopy techniques to probe tissue oxygenation and tissue oxygenation kinetics has led to the need for further quantitation of spectroscopic signals. In this paper, we briefly review the theory of light transport in strongly scattering media as monitored in the time and frequency domains, and use this theory to develop algorithms for quantitation of hemoglobin saturation from the photon decay rate (delta log R/delta t) obtained using time-resolved spectroscopy, and from the phase-shift (theta) obtained from frequency-resolved, phase-modulated spectroscopy. To test the relationship of these optical parameters, we studied the behavior of delta log R/delta t and theta as a function of oxygenation in model systems which mimicked the optical properties of tissue. Our results show that deoxygenation at varying hemoglobin concentrations can be monitored with the change in the photon decay kinetics, delta delta log R/delta t in the time-resolved measurements, and with the change in phase-shift, delta theta, in the frequency-resolved technique. Optical spectra of the adult human brain obtained with these two techniques show similar characteristics identified from the model systems.     

Fiber background rejection and crystal over-response compensation for GaN based in vivo dosimetry

For dosimetric measurements using an implantable optical fiber probe with GaN (Gallium Nitride) scintillator as radioluminescence (RL) transducer, a bi-channel method is proposed to reject the background contribution of the irradiated fiber segment. It is based on spectral differences between the narrow-band light emission from GaN and the large-band background from the irradiated optical fiber. Experimental validation of this method using 6 MV photon beam has shown that the remaining background contribution after subtraction is below 1.2% for square field sizes ranging from 3 cm to 20 cm. Furthermore, a compensation method for the over-response of GaN is also proposed, since GaN is not tissue equivalent. The over-response factor of GaN exhibits a linear increase with square field aperture and depends on depth from phantom surface. This behaviour is modelled to allow compensation in specific conditions. The proposed method has been evaluated and has shown a maximum deviation of 3% for a 6 MV photon beam and 1% for an 18 MV photon beam at a depth beyond the build-up region.